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Abstract:

A method of determining the immune response of a mammal to circulating
tumour marker proteins is described in which a sample of bodily fluid,
for example plasma or serum, is contacted with a panel of two or more
distinct tumour marker antigen. The presence of complexes between the
tumour marker antigens and any autoantibodies to the antigens present in
the sample are detected and provide an indication of an immune response
to a circulating tumour marker protein. The method is useful for the
diagnosis of cancer, particularly for identifying new or recurrent cancer
in an otherwise assymptomatic patient.

Claims:

1-3. (canceled)

4. A method of detecting the immune response of a mammal to circulating
tumour marker proteins or tumour cells expressing the tumour marker
proteins comprising: (a) contacting a sample of bodily fluids from the
mammal with a panel of two or more distinct tumour marker antigens;
wherein at least one of the two or more tumour marker antigens is
selected from the group consisting of MUC1, p53, c-erbB2, Ras, c-myc,
BRCA1, BRCA2, PSA, APC and CA125 (b) determining the presence or absence
of complexes of the tumour marker antigens bound to autoantibodies
present in the sample of bodily fluids, the autoantibodies being
immunologically specific to the tumour marker proteins; whereby the
presence of the complexes is indicative of the immune response to
circulating tumour marker proteins or tumour cells expressing the tumour
marker proteins.

5. A method of detecting the immune response of a mammal to circulating
tumour marker proteins or tumour cells expressing the tumour marker
proteins: (a) contacting a sample of bodily fluids from the mammal with a
panel of two or more distinct tumour marker antigens; (b) determining the
presence or absence of complexes of the tumour marker antigens bound to
autoantibodies present in the sample of bodily fluids, the autoantibodies
being immunologically specific to the tumour marker proteins; whereby the
presence of the complexes is indicative of the immune response to
circulating tumour marker proteins or tumour cells expressing the tumour
marker proteins wherein the complexes detected are indicative of cancer.

6. The method of claim 4 wherein the panel comprises p53 and c-erbB2.

7. The method of claim 5 wherein the cancer is bladder cancer and the
panel is selected from at least two tumour marker antigens selected from
the group consisting of p53, c-erbB2, MUC1 and c-myc.

8-9. (canceled)

10. The method of claim 6 wherein the complexes detected are indicative
of cancer, the cancer is breast cancer, and the panel also includes at
least one tumour marker antigen selected from the group consisting of
MUC1, c-myc, BRCA1, BRCA2, and PSA.

11. (canceled)

12. The method of claim 5 wherein the cancer is colorectal cancer and the
panel is selected from at least two tumour marker antigens selected from
the group consisting of p53, Ras, c-erbB2 and APC.

13. The method of claim 5 wherein the cancer is prostate cancer and the
panel is selected from at least two tumour marker antigens selected from
the group consisting of p53, PSA, BRCA1 and c-erbB2.

14. The method of claim 5 wherein the cancer is ovarian cancer and the
panel is selected from at least two tumour marker antigens selected from
the group consisting of p53, CA125, c-erbB2 and BRCA1.

15. The method of claim 5 wherein the cancer is breast cancer and the
panel is selected from at least two tumour marker antigens selected from
the group consisting of p53, MUC1, c-erbB2, c-myc, BRCA1, BRCA2 and PSA.

16-18. (canceled)

19. A method for the detection of cancer comprising: (a) contacting a
sample of bodily fluids from a mammal with a panel of two or more
distinct tumour marker antigens; (b) determining the presence or absence
of complexes of the tumour marker antigens bound to autoantibodies
present in the sample of bodily fluids, the autoantibodies being
immunologically specific to the tumor marker proteins; wherein the
presence of the complexes is indicative of cancer, and wherein the cancer
is recurrent disease in a patient previously diagnosed as carrying tumour
cells, wherein the patient has undergone treatment to reduce the number
of the tumour cells.

20-28. (canceled)

29. A method of determining the immune response of a patient to two or
more circulating tumour marker proteins or to tumour cells expressing the
tumour marker proteins and identifying which one of the two or more
tumour marker proteins elicits the strongest immune response in the
patient, comprising: (a) contacting a sample of bodily fluids from the
patient with a panel of two or more distinct tumour marker antigens; (b)
measuring the amount of complexes formed by binding of each of the tumour
marker antigens to autoantibodies present in the sample of bodily fluids,
the autoantibodies being immunologically specific to the tumour marker
proteins; whereby the presence of the complexes is indicative of the
immune response to circulating tumour marker proteins or tumour cells
expressing the tumour marker proteins, wherein the measurement obtained
acts as an indicator of the relative strength of the immune response to
each tumour marker protein and thereby identifies which one of the tumour
marker proteins elicits the strongest immune response in the patient
wherein at least one of the tumour marker antigens is selected from the
group consisting of MUC1, c-erbB2, c-myc, Ras, p53, BRCA1, BRCA2, PSA,
APC or CA125.

30. The method of claim 26 wherein the relative strength of the immune
response to each of the tumour marker proteins or tumor cells indicates
selection of a course of anti-cancer treatment.

31. The method of claim 30 wherein one or more tumour marker proteins
identified as eliciting a strong immune response in the patient indicate
selection of the course of the anti-cancer treatment.

32-41. (canceled)

42. A method of detecting the immune response of a mammal to circulating
tumour marker proteins or tumour cells expressing the tumour marker
proteins comprising: (a) contacting a sample of bodily fluids from said
mammal with a panel of two or more distinct tumour marker antigens; (b)
determining the presence or absence of complexes of the tumour marker
antigens bound to autoantibodies present in the sample of bodily fluids,
the autoantibodies being immunologically specific to the tumour marker
proteins; whereby the presence of the complexes is indicative of the
immune response to circulating tumour marker proteins or tumour cells
expressing the tumour marker proteins further comprising quantifying the
immune response of a mammal to circulating tumour marker proteins or
tumour cells expressing the tumour marker proteins wherein at least one
of the tumour marker proteins is selected from the group consisting of
c-erbB2, Ras, c-myc, p53, BRCA1, BRCA2, APC, PSA and CA125, and wherein
the method further comprises measuring the quantity of complexes formed
by binding of at least one tumour marker antigen to autoantibodies
present in the sample of bodily fluids, the autoantibodies being
immunologically specific to the tumour marker protein; and wherein the
measurement of the quantity of complexes indicates the amount of the
autoantibodies present in the sample.

43-45. (canceled)

46. A method for the detection of cancer comprising: (a) contacting a
sample of bodily fluids with a panel of two or more tumor marker antigens
selected from the group consisting of c-erbB2, ras, biotinylated c-myc,
BRCA1, BRCA2, APC, PSA, CA125 and biotinylated p53; (b) measuring the
quantity of complexes formed by binding of at least one tumour marker
antigen to autoantibodies present in the sample of bodily fluids, the
autoantibodies being immunologically specific to the tumour marker
protein; and (c) using the measurement obtained in (b) as a indicator of
the amount of the autoantibodies present in the sample.

47. (canceled)

48. A method for the detection of cancer, wherein the cancer is early
neoplastic or early carcinogenic change in asymptomatic patients
comprising: (a) contacting a sample of bodily fluids with at least one
tumour marker antigen selected from the group consisting of c-erbB2, ras,
biotinylated c-myc, BRCA1, BRCA2, APC, PSA, CA125 and biotinylated p53;
(b) measuring the quantity of complexes formed by binding of at least one
tumour marker antigen to autoantibodies present in the sample of bodily
fluids, the autoantibodies being immunologically specific to the tumour
marker protein; and (c) using the measurement obtained in (b) as a
indicator of the amount of the autoantibodies present in the sample,
wherein the cancer is recurrent disease in a patient previously diagnosed
as carrying tumour cells, and wherein the patient has undergone treatment
to reduce the number of the tumour cells.

49. The method of claim 46 wherein the cancer is recurrent disease in a
patient previously diagnosed as carrying tumour cells, wherein the
patient has undergone treatment to reduce the number of the tumour cells.

50. The method of claim 49 wherein the amount of autoantibodies present
monitors the progress of neoplastic disease.

51. The method of claim 42 wherein the amount of autoantibodies present
identifies those individuals who are at increased risk of developing
cancer in a population of asymptomatic individuals.

52. (canceled)

53. The method of claim 49 wherein the amount of autoantibodies present
predicts the response of the patient with cancer to anti-cancer
treatment.

55. A method of detecting the immune response of a mammal to a
circulating tumour marker proteins or tumour cells expressing the tumour
marker proteins wherein the tumour marker proteins are MUC1, p53,
c-erbB2, Ras, c-myc, BRCA1, BRCA2, PSA, APC or CA125, the method
comprising the steps of: (a) contacting a sample of bodily fluids from
the mammal with MUC1, c-erbB2, Ras, biotinylated c-myc, biotinylated p53,
BRCA1, BRCA2, APC, PSA or CA125 or an antigenic fragment thereof, and;
(b) determining the presence or absence of complexes of the tumour marker
protein or antigenic fragment thereof bound to autoantibodies present in
the sample of bodily fluids, the autoantibodies being immunologically
specific to the tumour marker protein or antigenic fragment thereof;
whereby the presence of the complexes is indicative of the immune
response to the circulating tumour marker protein or tumour cells
expressing the tumour marker protein.

56-57. (canceled)

58. The method of claim 55 wherein the presence of complexes indicates
the presence of cancer.

59. (canceled)

60. The method of claim 58 wherein the cancer is early neoplastic or
early carcinogenic change in asymptomatic patients.

61. The method of claim 58 wherein the cancer is recurrent disease in a
patient previously diagnosed as carrying tumour cells, wherein the
patient has undergone treatment to reduce the number of the tumour cells.

62. The method of claim 58 wherein the presence of complexes indicates
the progress of cancer or other neoplastic disease.

63. The method of claim 58 wherein the presence of complexes identifies
those individuals who are at increased risk of developing cancer in a
population of asymptomatic individuals.

64-67. (canceled)

68. A method of detecting recurrent disease in a patient previously
diagnosed as carrying tumour cells, wherein the patient has undergone
treatment to reduce the number of the tumour cells, comprising: (a)
contacting a sample of bodily fluids from the patient with MUC1 protein
or an antigenic fragment thereof wherein the MUC1 manifests all the
antigenic characteristics of a MUC1 protein obtainable from the bodily
fluids of a patient with advanced breast cancer; (b) determining the
presence or absence of complexes of the MUC1 protein or antigenic
fragment thereof bound to autoantibodies present in the sample of sample
of bodily fluids, the autoantibodies being immunologically specific to
MUC1; wherein the presence of the complexes indicates the presence of
recurrent disease in the patient.

69. The method of claim 68 wherein the patient has undergone primary
breast cancer treatment to reduce the number of the tumor cells.

70-72. (canceled)

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. patent
application Ser. No. 11/953,237, which is a continuation application of
U.S. patent application Ser. No. 09/700,092, filed May 16, 2001, which is
a national stage filing under 35 U.S.C. §371 of PCT International
Application PCT/GB99/01479, filed May 11, 1999, which claims priority to
Great Britain Application No. 9810040.7, filed May 11, 1998, all of which
are incorporated herein by reference.

[0002] The invention relates to methods of detecting or quantitatively
measuring the immune response of a mammal to circulating tumour markers
or tumour markers expressed on the surface of tumour cells, also to
tumour marker antigens for use in these methods, to kits for performing
the methods and to the use of these methods in the detection of cancer,
in monitoring the progress of cancer, in detecting recurrent disease in
cancer patients who have previously undergone anti-cancer treatment and
in predicting the response of a cancer patient to a particular course of
treatment.

[0003] The development and progression of cancer in a patient is generally
found to be associated with the presence of markers in the bodily fluid
of the patient, these "tumour markers" reflecting different aspects of
the biology of the cancer (see Fateh-Maghadam, A. & Steilber, P. (1993)
Sensible use of tumour markers. Published by Verlag GMBH, ISBN
3-926725-07-9). Tumour markers are often found to be altered forms of the
wild type proteins expressed by `normal` cells, in which case the
alteration may be a change in primary amino acid sequence, a change in
secondary, tertiary or quaternary structure or a change in
post-translational modification, for example, abnormal glycosylation.
Alternatively, wild type proteins which are up-regulated or
over-expressed in tumour cells, possibly as a result of gene
amplification or abnormal transcriptional regulation, may also be tumour
markers.

[0004] Established assays for tumour markers present in bodily fluids tend
to focus on the detection of tumour markers which reflect tumour bulk and
as such are of value late in the disease process, for example, in the
diagnosis of metastatic disease. The most widely used of these markers
include carcinoembryonic antigen (CEA) and the glycoprotein termed CA
15.3, both of which have been useful mainly as indicators of systemic
disease burden and of relapse following therapy (Molina, R., Zanon, G.,
Filella, X. et al. Use of serial carcinoembryonic antigen and CA 15.3
assays in detecting relapses in breast cancer patients. (1995) Breast
Cancer Res Treat 36: 41-48) These markers are of limited use earlier in
the disease progression, for example in the screening of asymptomatic
patients. Thus, in the search for tumour markers present in bodily fluid
that are of use earlier in the disease process the present inventors have
sought to identify markers which do not depend on tumour bulk per se.

[0005] Differences between a wild type protein expressed by `normal` cells
and a corresponding tumour marker protein may, in some instances, lead to
the tumour marker protein being recognised by an individual's immune
system as `non-self` and thus eliciting an immune response in that
individual. This may be a humoral (i.e B cell-mediated) immune response
leading to the production of autoantibodies immunologically specific to
the tumour marker protein. Autoantibodies are naturally occurring
antibodies directed to an antigen which an individual's immune system
recognises as foreign even though that antigen actually originated in the
individual. They may be present in the circulation as circulating free
autoantibodies or in the form of circulating immune complexes consisting
of autoantibodies bound to their target tumour marker protein.

[0006] As an alternative to the direct measurement or detection of tumour
marker protein in bodily fluids, assays could be developed to measure the
immune response of the individual to the presence of tumour marker
protein in terms of autoantibody production. Such assays would
essentially constitute indirect detection of the presence of tumour
marker protein. Because of the nature of the immune response, it is
likely that autoantibodies can be elicited by a very small amount of
circulating tumour marker protein and indirect methods which rely on
detecting the immune response to tumour markers will consequently be more
sensitive than methods for the direct measurement of tumour markers in
bodily fluids. Assay methods based on the detection of autoantibodies may
therefore be of particular value early in the disease process and
possibly also in relation to screening of asymptomatic patients, for
example to identify individuals "at risk" of developing disease.

[0007] Tumour marker proteins observed to elicit serum autoantibodies
include a particular class of mutant p53 protein, described in U.S. Pat.
No. 5,652,115, which can be defined by its ability to bind to the 70 kd
heat shock protein (hsp70). p53 autoantibodies can be detected in
patients with a number of different benign and malignant conditions
(described in U.S. Pat. No. 5,652,115) but are in each case present in
only a subset of patients. For example, one study utilizing an ELISA
assay for detection of autoantibodies directed against the p53 protein in
the serum of breast cancer patients reported that p53 autoantibodies were
produced by 26% of patients and 1.3% of control subjects (Mudenda, B.,
Green, J. A., Green, B. et al. The relationship between serum p53
autoantibodies and characteristics of human breast cancer. (1994) Br J
Cancer 69: 4445-4449.). A second tumour marker protein known to elicit
serum autoantibodies is the epithelial mucin MUC1 (Hinoda, Y. et al.
(1993) Immunol Lett. 35: 163-168; Kotera, Y. et al. (1994) Cancer Res.
54: 2856-2860).

[0008] In most cancers resulting from a progressive accumulation of
genetic alterations, such as breast cancer, the presence of tumour
markers in bodily fluids reflects the development and progression of
disease but no single marker on its own summates all clinically important
parameters. For example, the characteristics of a marker useful for
diagnosis of cancer may be quite different from markers which convey
information about prognosis. Furthermore, in each clinical situation
(i.e. diagnosis or prognosis) different markers may be required when
dealing with primary cancer and secondary (metastatic) cancer and a
different marker again may be required to provide a method of measuring
the effectiveness of a particular course of treatment. Different clinical
situations therefore require different biological markers and, as has
been observed with p53, not all patients express the same set of tumour
marker proteins. It is therefore difficult to envisage any one single
tumour marker being universally applicable to all patients in all stages
of disease.

[0009] It is an object of the present invention to provide an improved
assay system for the detection of bodily fluids-borne tumour markers
which is more generally useful in all patients and in a variety of
different clinical situations.

[0010] Accordingly, in a first aspect the invention provides a method of
detecting the immune response of a mammal to circulating tumour marker
proteins or tumour cells expressing said tumour marker proteins, which
method comprises steps of: [0011] (a) contacting a sample of bodily
fluids from said mammal with a panel of two or more distinct tumour
marker antigens; [0012] (b) determining the presence or absence of
complexes of said tumour marker antigens bound to autoantibodies present
in said sample of bodily fluids, said autoantibodies being
immunologically specific to said tumour marker proteins. whereby the
presence of said complexes is indicative of the immune response to
circulating tumour marker proteins or tumour cells expressing said tumour
marker proteins.

[0013] The method of the invention, which may be hereinafter referred to
as a `panel assay`, utilises a panel of two or more tumour marker
antigens to monitor the overall immune response of an individual to a
tumour or other carcinogenic/neoplastic change. The method thus provides
essentially a `profile` of the immune response for that individual,
indicating which tumour markers elicit an immune response resulting in
autoantibody production. The method of the invention is preferred for the
detection of an immune response resulting in the production of
circulating free autoantibodies.

[0014] Because the assay method of the invention performed on a sample of
bodily fluids taken from the patient it is essentially non-invasive and
can be repeated as often as is thought necessary to build up a profile of
the patient's immune response throughout the course of disease. As used
herein the term `bodily fluids` includes plasma, serum, whole blood,
urine, sweat, lymph, faeces, cerebrospinal fluid or nipple aspirate. The
type of bodily fluid used may vary depending upon the type of cancer
involved and the use that the assay is being put to. In general, it is
preferred to perform the method on samples of serum or plasma.

[0015] As will be illustrated in the Examples given below, the use of a
panel of two or more tumour marker antigens to monitor autoantibody
production is more sensitive than the use of single markers and gives a
much lower frequency of false negative results. The actual steps of
detecting autoantibodies in a sample of bodily fluids may be performed in
accordance with immunological assay techniques known per se in the art.
Examples of suitable techniques include ELISA, radioimmunoassays and the
like. In general terms, such assays use an antigen which may be
immobilised on a solid support. A sample to be tested is brought into
contact with the antigen and if autoantibodies specific to the tumour
marker protein are present in the sample they will immunologically react
with the antigen to form autoantibody-antigen complexes which may then be
detected or quantitatively measured. Detection of autoantibody-antigen
complexes is preferably carried out using a secondary anti-human
immunoglobulin antibody, typically anti-IgG or anti-human IgM, which
recognise general features common to all human IgGs or IgMs,
respectively. The secondary antibody is usually conjugated to an enzyme
such as, for example, horseradish peroxidase (HRP) so that detection of
autoantibody/antigen/secondary antibody complexes is achieved by the
addition of an enzyme substrate and subsequent colorimetric,
chemiluminescent or fluorescent detection of the enzymatic reaction
products.

[0016] The panel assay of the invention uses a panel of tumour
marker-related antigens. The panel may be tailored to detect a particular
cancer, or a cancer at a particular stage of development. The tumour
marker antigens may be wild type or mutant tumour marker proteins
isolated from samples of biological fluid from normal individuals or from
cancer patients or from cell lines expressing the tumour marker protein
or they may be full length recombinant tumour marker proteins, viral
oncogenic forms of tumour marker proteins or antigenic fragments of any
of the aforementioned proteins. The term `antigenic fragment` as used
herein means a fragment which is capable of eliciting an immune response.

[0017] The panel assay may be performed in a multi-well format in which
each one of the two or more antigens is placed in a separate well of a
multi-well assay plate or, alternatively, in a single-pot format in which
the entire panel of antigens is placed in a single container. The panel
assay may be performed in a qualitative format in which the objective is
simply detection of the presence or absence of autoantibodies or in a
quantitative format which provides a quantitative measurement of the
amount of autoantibodies present in a sample.

[0019] Preferably the tumour marker antigens are labelled with biotin so
that they can easily be attached to a solid support, such as a multi-well
assay plate, by means of the biotin/avidin or biotin/streptavidin
interaction. Tumour marker antigens labelled with biotin may be referred
to herein as `biotinylated` proteins. To facilitate the production of
biotinylated tumour marker antigens for use in the assay methods of the
invention, cDNAs encoding a full length recombinant tumour marker
protein, a truncated version thereof or an antigenic fragment thereof may
be expressed as a fusion protein labelled with a protein or polypeptide
tag to which the biotin co-factor may be attached via an enzymatic
reaction. A useful system for the expression of biotinylated fusion
proteins is the PinPoint® system supplied by Promega Corporation,
Madison Wis., USA. The present inventors have surprisingly found that
with the use of biotinylated tumour marker antigens as antigens they are
able to detect autoantibodies in a much higher percentage of patients
than is observed using non-biotinylated antigen.

[0020] The assay method of the invention may be employed in a variety of
different clinical situations such as, for example, in the detection of
primary or secondary (metastatic) cancer, in screening for early
neoplastic or early carcinogenic change in asymptomatic patients or
identification of individuals `at risk` of developing cancer
(particularly breast cancer, bladder cancer, colorectal cancer or
prostate cancer) in a population or asymptomatic individuals, in the
detection of recurrent disease in a patient previously diagnosed as
carrying tumour cells who has undergone treatment to reduce the number of
tumour cells or in predicting the response of an individual with cancer
to a course of anti-cancer treatment.

[0021] The assay method of the invention is suitable for detection of many
different types of cancer, of which examples are breast, bladder,
colorectal, prostate and ovarian. The assay of the invention may
complement existing methods of screening and surveillance. For example in
the case of primary breast cancer it could be used to alert clinicians to
biopsy small lesions on mammograms which radiographically do not appear
suspicious or to carry out breast imaging or to repeat imaging earlier
than planned. In the clinic, the assay method of the invention is
expected to be more objective and reproducible compared to current
imaging techniques (i.e. mammography and ultrasound), the success of
which can be operator-dependent.

[0031] In the case of colorectal cancer suitable panels could be selected
from the following: [0032] p53 and ras with optional c-erbB2 and/or
APC; [0033] p53 and APC with optional c-erbB2 and/or Ras; Ras and APC
with optional p53 and/or c-erbB2

[0034] In the case of prostate cancer suitable panels could be selected
from the following: [0035] p53 and PSA with optional BRCA1 and/or
c-erbB2; c-erbB2 and PSA with optional p53 and/or BRCA1.

[0036] In the case of ovarian cancer suitable panels could be selected
from the following: [0037] p53 and CA125 with optional c-erbB2 and/or
BRCA1; c-erbB2 and CA125 with optional p53 and/or BRCA1.

[0038] In a second aspect the invention provides a method of determining
the immune response of a patient to two or more circulating tumour marker
proteins or to tumour cells expressing said tumour marker proteins and
identifying which one of said two or more tumour marker proteins elicits
the strongest immune response in the patient, the method comprising
contacting a sample of bodily fluids from said patient with a panel of
two or more distinct tumour marker antigens, measuring the amount of
complexes formed by binding of each of said tumour marker antigens to
autoantibodies present in the sample of bodily fluids, said
autoantibodies being immunologically specific to said tumour marker
proteins and using the measurement obtained as an indicator of the
relative strength of the immune response to each tumour marker protein
and thereby identifying which one of said two or more tumour marker
proteins elicits the strongest immune response in the patient.

[0039] The assay described above, which may be hereinafter referred to as
a `selection assay` is useful in the selection of a course of vaccine
treatment wherein the single tumour marker protein identified as
eliciting the strongest immune response or a combination of markers
eliciting strong immune response is/are used as the basis of an
anti-cancer vaccine treatment.

[0040] Preferred tumour marker antigens for use in the selection assay are
any of the tumour marker antigens mentioned above and preferably the
antigens are labelled with biotin. The actual steps of detecting
autoantibodies in a sample of bodily fluids may be performed in
accordance with known immunological assay techniques, as described above
for the panel assay.

[0041] The invention also provides methods for the detection or
quantitative measurement of the immune response of a mammal to a
circulating tumour marker protein or tumour cells expressing the tumour
marker protein wherein the tumour marker protein is MUC1, c-erbB2, Ras,
c-myc, BRCA1, BRCA2, PSA, APC, CA125 or p53, the method comprising the
steps of contacting a sample of bodily fluids from the mammal with the
tumour marker antigen and determining the presence or absence of
complexes of the tumour marker antigen bound to autoantibodies
immunologically specific to the tumour marker protein or antigenic
fragment thereof, whereby the presence of said complexes is indicative of
the immune response to said circulating tumour marker protein or tumour
cells expressing the tumour marker protein.

[0042] The assays described above, which may be hereinafter referred to as
`single marker assays`, use a single type of tumour marker as antigen
rather than using a panel of two or more tumour markers. The single
marker assays may be used in any clinical situation, for example,
screening for early neoplastic or carcinogenic change in asymptomatic
patients, identification of individuals `at risk` of developing cancer,
early diagnosis and early detection of recurrence in a patient previously
diagnosed as carrying tumour cells which patient has undergone treatment
to reduce the number of said tumour cells or in predicting the response
of a patient to a course of anti-cancer treatment, including surgery,
radiotherapy, immune therapy, vaccination etc.

[0043] The single marker assays are particularly useful in situations
where the tumour marker eliciting the strongest immune response in a
given patient has been previously identified, possibly using the
selection assay described above. For example, in a situation in which an
initial selection assay has been performed to establish which tumour
marker elicits the strongest immune response in a given patient,
subsequent follow-up, detection of recurrence or monitoring of treatment
may be carried out using a single marker assay to only detect or measure
autoantibodies to that tumour marker previously identified as eliciting a
strong immune response in that patient.

[0044] The actual steps of detecting autoantibodies in a sample of bodily
fluids may be performed in accordance with known immunological assay
techniques, as described above for the panel assay. Preferably the tumour
marker protein used as antigen is labelled with biotin so that it may be
easily attached to a solid support by means of the biotin/avidin or
biotin/streptavidin interaction.

[0045] In a further aspect, the present invention provides a preparation
comprising a human MUC1 protein which MUC1 protein manifests all the
antigenic characteristics of a MUC1 protein obtainable from the bodily
fluids of a patient with advanced breast cancer.

[0046] Preferably the MUC1 protein exhibits altered affinity for the
antibodies B55, C595, BC4W154, DF3, B27.29, 115D8, 27.1, SM3, Ma552, HMPV
and BC2 compared to MUC1 protein isolated from normal human urine. Most
preferably the MUC1 protein is isolated from the serum of one or more
human patients with advanced breast cancer. This can be accomplished
using the protocol given in the Examples listed herein.

[0047] As will be described in detail in Example 2, the present inventors
have found immunological differences between MUC1 isolated from normal
individuals and MUC1 isolated from patients with advanced breast cancer.
Possibly as a result of these differences, the inventors have found that
the MUC1 protein isolated from serum of patients with advanced breast
cancer (hereinafter referred to as ABC MUC1) is more sensitive when used
as antigen in an assay to detect autoantibodies specific to MUC1 than
either MUC1 isolated from urine of normal individuals, synthetic MUC1 or
MUC1 isolated from a range of different cultured cells. MUC1 isolated
from the serum of patients with advanced breast cancer is therefore
preferred for use as antigen in the panel assay method and the single
marker assay methods described herein.

[0048] MUC1 has recently attracted interest as a target for immunotherapy
of adenocarcinomas and several Phase I clinical trials involving
different MUC1 vaccine substrates, adjuvants and carrier proteins have
been carried out (Goydos, J. S. et al. (1996) J Surgical Res. 63:
298-304; Xing, P. X. et al. (1995) Int. J Oncol. 6: 1283-1289; Reddish,
M. A. et al. (1996) Cancer Immunol. Immunother. 42: 303-309; Graham, R.
A. et al. (1996) Cancer Immunol. Immunother. 42: 71-80). Methods for the
detection of anti-MUC1 autoantibodies using MUC1 isolated from the serum
of patients with advanced breast cancer as antigen will be of particular
use in monitoring the success of MUC1 vaccine therapy. In this case the
aim of the assay will be to detect anti-MUC1 antibodies produced in
response to the vaccine rather than autoantibodies i.e. antibodies
produced in response to an exogenous antigen introduced into the body by
vaccination. Methods for the detection of autoantibodies directed to
other tumour markers would also be of use in monitoring the success of
vaccine therapy using the relevant tumour marker. For example, following
vaccination with a p53 antigenic preparation, the presence of anti-p53
antibodies could be monitored using the assay based on the use of
biotinylated p53 antigen described in the examples given below. Moreover,
the panel assay method could also be used in monitoring the success of
vaccine therapy, for example, in a situation where an individual has been
vaccinated with an antigenic preparation designed to elicit antibodies to
two or more different tumour markers.

[0049] In a still further aspect the invention provides a method of
detecting recurrent disease in a patient previously diagnosed as carrying
tumour cells, which patient has undergone treatment to reduce the number
of said tumour cells, which method comprises steps of contacting a sample
of bodily fluids from the patient with MUC1 protein or an antigenic
fragment thereof, determining the presence or absence of complexes of
said MUC1 protein or antigenic fragment thereof bound to autoantibodies
present in said sample of bodily fluids, said autoantibodies being
immunologically specific to MUC1, whereby the presence of said complexes
indicates the presence of recurrent disease in said patient.

[0050] The method described above may be repeated on a number of occasions
to provide continued monitoring for recurrence of disease. The method is
particularly preferred for the monitoring of patients previously
diagnosed with primary breast cancer, colorectal cancer, prostate cancer
or bladder cancer, which patients have undergone treatment (e.g. surgery)
to remove or reduce the size of their tumour. In this instance, the
presence of anti-MUC1 autoantibodies in the patient's serum after
treatment may be indicative of recurrence of disease.

[0051] Also provided by the invention are assay kits suitable for
performing the methods for the detection of autoantibodies described
herein. Such kits include, at least, samples of the tumour marker
antigens to be used as antigen in the assay and means for contacting the
sample to be tested with a sample of the antigen.

[0052] The contents of all documents, articles and references cited herein
are incorporated herein by reference.

[0053] The present invention will be further understood with reference to
the following Examples and the accompanying Figures in which:

[0054] FIG. 1: shows the results of assays for autoantibodies to MUC1, p53
and c-erbB2 in samples of serum taken from 21 patients diagnosed with
primary breast cancer. Panel A: anti-p53 autoantibodies; Panel B:
anti-c-erbB2 autoantibodies and Panel C: anti-MUC1 autoantibodies. In
each case, the dotted line represents the cut-off value for normality.

[0056] FIG. 3: shows continuous monitoring for recurrent disease in three
post-operative breast cancer patients. Quantitative assays for anti-MUC1,
anti-cerbB2 and anti-p53 autoantibodies and for the tumour marker CA15-3
(®) were performed on samples of serum taken at two or three monthly
intervals post-surgery.

[0057] FIG. 4: shows the range of autoantibody levels found in assays for
autoantibodies to c-erbB2, c-myc, MUC1 and p53 in normal individuals and
patients with early primary breast cancer (PBC).

[0058] FIG. 5: summarises the detection rate for primary breast cancer in
an analysis of autoantibody levels in a series of healthy controls and
patients with primary breast cancer, PBC subdivided by Stage 1--i.e.
lymph node negative and Stage 2--i.e. lymph node positive and patients
with metastatic cancer at 100% confidence.

[0059] FIG. 6: summarises the detection rate for primary breast cancer in
an analysis of autoantibody levels in a series of healthy controls and
patients with PBC subdivided by Stage 1--i.e. lymph node negative and
Stage 2--i.e. lymph node positive and patients with metastatic cancer at
95% confidence.

[0060] FIG. 7: shows the sensitivity for primary breast cancer in an
analysis of autoantibody levels in a series of healthy controls and
patients with Stage 1 or Stage 2 primary breast cancer at 95% confidence.

[0061] FIG. 8: shows the levels of autoantibodies to MUC1, p53 and c-erbB2
in the serum of three patients previously diagnosed with breast cancer
measured sequentially during follow-up until the patient manifested
recurrent disease.

[0062] FIG. 9: shows the autoantibody levels in further samples from the
second patient in FIG. 10 (AEC at 36 months) taken up to recurrence and
during treatment for recurrence. Sequential measurements of established
tumour markers reflecting tumour bulk (e.g. CA15-3 and CEA) were within
the normal range throughout this period (data not shown).

[0063] FIG. 10: shows follow-up autoantibody levels in post-operative
serum samples from two patients, one who did not develop recurrent
disease (no REC) and the other who did (REC at 36 months).

[0064] FIG. 11: summarises the detection rates in an analysis of
autoantibody levels (p53, MUC1, c-erbB2 and c-myc) in samples of serum
taken from patients with urologically benign disorders and various stages
of bladder cancer.

[0065] * indicates patients which were benign with respect to urology
(i.e. did not have a urological malignancy), but six cases (all with
positive autoantibody status) had evidence of other malignancies.

[0067] FIG. 12: summarises the detection rate for colorectal cancer in an
analysis of autoantibody levels in the serum of healthy controls,
patients with colonic polyps and patients with colorectal cancer at 100%
confidence compared to a pre-defined group of healthy controls.

[0068] FIG. 13: summarises the detection rate for colorectal cancer in an
analysis of autoantibody levels serum of healthy controls, patients with
colonic polyps and patients with colorectal cancer at 95% confidence
compared to a pre-defined group of healthy controls.

[0069] FIG. 14: summarises the detection rate in an analysis of
autoantibody levels in the serum of healthy controls, patients with
primary breast cancer and asymptomatic women known to be BRCA1 mutant
carriers at 100% confidence compared to a pre-defined group of healthy
controls.

[0070] FIG. 15: summarises the detection rate for prostate cancer in an
analysis of autoantibody levels in the serum of healthy controls and
patients with prostate cancer at 95% confidence compared to a pre-defined
group of healthy controls.

EXAMPLES

Example 1

Isolation of ABC MUC1 from Advanced Breast Cancer Patients

Method

[0071] ABC MUC1 was purified from pooled sera taken from 20 patients with
advanced breast cancer using immunoaffinity chromatography as follows:

[0072] The mouse monoclonal anti-MUC1 antibody B55 (also known as NCRC 11
and described by Ellis et al. (1984) Histopathology. 8: 501-516 and in
International patent application No. WO 89/01153) was conjugated to
CNBrsepharose beads. Pooled sera from patients diagnosed with advanced
breast cancer was diluted 1/10 in phosphate buffered saline (PBS) and
then incubated with the antibody conjugated sepharose beads (25 ml
diluted sera to 1 ml packed volume of beads) overnight at 4° C.
with rolling. The beads were then packed by centrifugation and the
supernatant removed. In order to wash away unbound serum components the
beads were resuspended in PBS, rolled for 10 minutes, packed by
centrifugation and the supernatant removed. This washing sequence was
repeated 5 times (or until A280 nm of the supernatant was -0). The washed
beads were then resuspended in 0.25M glycine pH 2.5, rolled at room
temperature for 10 minutes, packed by centrifugation and the supernatant
removed. This supernatant was adjusted to pH 7 by the addition of Tris
and stored at 4° C. labelled `glycine fraction`. The beads were
then resuspended in 1 ml 25 mM diethylamine (DEA) pH11, rolled at room
temperature for 10 minutes, packed by centrifugation and the supernatant
removed. This supernatant was again adjusted to pH 7 by the addition of
Tris and stored at 4° C. labelled `25 DEA fraction`. The beads
were finally resuspended in 1 ml 100 mM DEA pH11, rolled at room
temperature for 10 minutes, packed by centrifugation and the supernatant
removed. The final supernatant was again adjusted to pH 7 by the addition
of Tris and stored at 4° C. labelled `100 DEA fraction`. The MUC1
content of the three fractions (glycine fraction, 25 DEA fraction and 100
DEA fraction) was confirmed by ELISA using the mouse monoclonal anti-MUC1
antibody C595 (commercially available from Serotec).

Example 2

Immunological Characterisation of ABC MUC1 Isolated from the Serum of
Patients with Advanced Breast Cancer

[0073] ABC MUC1 isolated from the serum at least 20 patients with advanced
breast cancer according to the procedure described in Example 1 can be
distinguished from MUC1 isolated from the urine of normal human subjects
(normal human urinary MUC1) on the basis of altered affinity for the
following mouse monoclonal anti-MUC1 antibodies:

[0074] Normal urinary MUC1 is available from Dr M. R. Price, Cancer
Research Laboratories, The University of Nottingham, University Park,
Nottingham. NG7 2RD, United Kingdom.

[0075] The affinity of each of the above antibodies for ABC MUC1, normal
human urinary MUC1 and also MUC1 protein purified from the human breast
cancer cell line ZR75-1 (purified from a tissue culture supernatant by
gel filtration) was measured by performing colorimetric ELISA assays
using each of the different antibodies and secondary anti-immunoglobulin
antibodies conjugated to HRP. Following addition of the colorimetric
substrate (TMB), measurements were taken of OD at 650 nm. The results of
the ELISA assays are presented graphically in FIG. 2. Values of Kd for
the binding of several of these antibodies to ABC MUC1 and normal human
urinary MUC1 are summarised in Table 1:

[0076] Commercially available cDNA for p53 (E. coli clone pBH53, deposited
in the American Type Culture Collection under accession number 79110) was
cloned into the PinPoint® plasmid vector (Promega Corporation, Madison
Wis., USA) using standard molecular biology techniques. The PinPoint®
vector is designed to facilitate the production of fusion proteins
comprising a biotinylation domain (consisting of a fragment of a biotin
carboxylase carrier protein) fused N-terminally to the target protein of
interest. Care was therefore taken during the cloning procedure to ensure
that the reading frame of p53 was maintained in the fusion protein.
Procedures for cloning in PinPoint® vectors are described in detail in
the Promega Protocols and Applications Guide obtainable from Promega
Corporation, Madison Wis., USA.

[0077] Fusion proteins expressed from the PinPoint® vector in E. coli
are biotinylated by an enzyme system of the E. coli host cells and may
therefore be purified or bound to an assay plate using conventional
avidin or streptavidin technology. For example, procedures for
purification of the fusion protein using avidin covalently attached to a
polymethacrylate resin are described in the Promega Protocols and
Applications Guide obtainable from Promega Corporation, Madison Wis.,
USA.

Example 4

Cloning of c-erbB2

Method

[0078] Full-length cDNA encoding c-erbB2 was cloned from the human breast
cancer cell line ZR75-1, which can be induced to up-regulate c-erbB2
expression by treatment with the anti-cancer drug tamoxifen.

[0079] Two T25 flasks of sub-confluent ZR75-1 cells (available from the
American Type Culture Collection and from the European Collection of Cell
Cultures, deposit number ATCC CRL1500) grown in RPMI plus 10% foetal calf
serum were induced to express c-erbB2 by 4 day stimulation with tamoxifen
at 7.5 pM (see Warri et al. (1996) Eur. J. Cancer. 32A: 134-140). The
cells were then harvested using trypsin/EDTA and washed three times with
PBS.

[0080] mRNA was extracted from the cell pellet using a Dynabead mRNA
purification kit according to the manufacturer's recommended protocol.
The mRNA was then used as a template for first strand cDNA synthesis
using the Pharmacia Ready-to-go® T primed first strand cDNA synthesis
kit. cDNA/mRNA was then blunt end ligated into the EcoRV site of the
PinPoint® vector. The ligation products were then transformed into Top
10 F E. coli cells (Invitrogen) following the manufacturer's supplied
protocol and the transformed cells grown overnight on LB agar plates
containing ampicillin. Colonies of the transformed E. coli were copied
onto nitrocellulose filter and then grown for 2 hours on LB agar
containing ampicillin and IPTG (1 mM). The colonies on the nitrocellulose
filter were fixed and lysed (15 minutes in the presence of chloroform
vapour followed by 18 hours in 100 mM Tris/HCL pH 7.8; 150 mM NaCl; 5 mM
MgCl2; 1.5% BSA; 1 μg/ml DNase 1; 40 μg/ml lysozyme).

[0087] Colonies identified as positive for c-erbB2 expression were picked
and grown up overnight in liquid culture of LB+ampicillin and small
amounts of plasmid DNA and protein were prepared from the culture for
analysis. Plasmids containing a c-erbB2 cDNA insert were identified using
restriction enzyme digestion and PCR using a primer pair specific to the
published c-erbB2 cDNA sequence, described by Yazici, H. et al. (1996)
Cancer Lett. 107: 235-239. DNA sequence analysis could then be used to
confirm 1) the presence of a c-erbB2 insert and 2) that the reading frame
of c-erbB2 is maintained in the resultant biotinylated fusion protein.
Protein samples prepared from E. coli cultures carrying a plasmid with a
c-erbB2 insert were analysed by SDS-PAGE and western blotting to ensure
that the correct protein was being expressed.

Example 5

Detection of the Immune Response of Patients with Primary Breast Cancer
Using a Panel Assay

Methods:

(A) Preparation of Biotinylated Antigen

[0088] E. coli transformed with the appropriate PinPoint® plasmid
expressing biotinylated antigen were grown in a 5 ml overnight culture
(LB+amp+biotin) and the overnight culture used to inoculate a 150 ml
culture. The 150 ml culture was grown to OD 0.4-0.6 then expression of
the fusion protein was induced by the addition of IPTG to a final
concentration of 1 mM and the induced culture incubated at 25° C.
The bacterial cells were harvested by centrifugation and then lysed by
gentle sonication in a Tris/EDTA buffer containing the protease inhibitor
PMSF. Cellular debris was removed by centrifugation at ˜50,000 g
and the resultant particle-free supernatant assayed by avidin ELISA to
confirm the presence of biotinylated protein.

(B) c-erbB2/p53 Autoantibody Assay Method [0089] 1. Standard 96 well
microtiter assay plates were coated with avidin, using 50 μl of a 1
μg/ml solution per well, and allowed to air dry overnight. The plates
were then washed once with PBS/Tween to remove residual salt crystals,
blocked for 60 minutes with a solution of 2% (w/v) PVP
(polyvinylpyrolidone 360) in PBS and washed three times using PBS/Tween.
[0090] 2. Particle free supernatant containing the appropriate
biotinylated antigen (prepared as described in section (1) above) was
plated out at 50 μl per avidin-coated well and then incubated for 60
minutes at room temperature with shaking to allow the biotin/avidin
binding reaction to take place. The plates were then washed four times
with PBS/Tween. [0091] 3. Serum samples to be tested for the presence of
autoantibodies (diluted 1/50 and 1/100 in PBS) were plated out in
triplicate (50 μl per well) and then incubated for 60 minutes with
shaking to allow formation of any autoantibody/antigen complexes. Plates
were then washed four times with PBS/Tween to remove unbound serum
components. [0092] 4. 50 μl of ARP conjugated anti-human IgG/IgM
antibody (obtained from Dako and used at a dilution recommended by the
manufacturer) was added to each well and incubated for 60 minutes at room
temperature with shaking. The plates were then washed again four times
with PBS/Tween. [0093] 5. 50 μl of TMB was added to each well and
measurements of OD at 650 nm for each well of the assay plate were taken
kinetically over a period of 10 minutes.

[0094] For each antigen, control assays were performed following the
procedure described above but using a sample of protein induced from E.
coli transformed with a control PinPoint® vector containing an
out-offrame cDNA instead of the particle free supernatant containing
biotinylated antigen. As it will be apparent to persons skilled in the
art, the above methodology can be adapted for use in the detection of
autoantibodies of any specificity with use of an appropriate biotinylated
antigen.

(C) MUC1 Autoantibody Assay

[0095] 1. ABC MUC1 isolated from the serum of patients with advanced
breast cancer according to the method of Example 1 (all three fractions
pooled) was diluted appropriately in PBS, plated out on a 96 well
microtiter assay plate at 50 μl per well and left to dry overnight.
The plate was then washed once with PBS/Tween to remove residual salt
crystals, blocked for 60 minutes using a solution of 2% (w/v) PVP in PBS
and washed three times with PBS/Tween. [0096] 2. Serum samples to be
tested for the presence of autoantibodies (diluted 1/50 and 1/100 in PBS)
were plated out in triplicate, adding 50 μl per well, and incubated
for 60 minutes at room temperature with shaking. The plate was then
washed four times with PBS/Tween. [0097] 3. 50 μl of HRP conjugated
anti-human IgG/IgM antibody (obtained from Dako and used at a dilution
recommended by the manufacturer) was added to each well and incubated for
60 minutes at room temperature with shaking. The plates were then washed
again four times with PBS/Tween. [0098] 4. 50 μl of TMB was added to
each well and measurements of OD at 650 nm for each well of the assay
plate were taken kinetically over a period of 10 minutes.

Results

[0099] Pre-operative blood samples taken from 21 patients diagnosed with
primary breast cancer were assayed for the presence of autoantibodies
against MUC1, p53 and c-erbB2. The results of these assays are shown in
FIG. 1 and summarised in Table 2.

[0100] FIG. 1 shows the results of the assays for autoantibodies specific
to MUC1, c-erbB2 and p53. For each set of data the dotted line represents
the cut-off value for normality. For the purposes of this study the
normal control patients were women who clinically and/or mammographically
had no evidence of breast cancer at the time of taking the serum sample.
In order to establish the cut-off value for normality, control assays
were performed on a total of 30 normal patients. Values below the dotted
line fall within the normal control range and were scored as negative (-)
in Table 2 whereas values above the dotted line were scored as positive
(+). Values which were difficult to score as negative or positive with a
reasonable degree of certainty were scored +/-. Patients scoring positive
in at least two of the assays were identified as strongly positive for
breast cancer (indicated "CANCER" in Table 2); patients scoring positive
in at least one of the assays were identified as probable for breast
cancer (indicated "cancer" in Table 2).

[0101] The results presented illustrate the predictive value of the three
autoantibody assays both when used individually and when used as a panel.
The use of a single assay to predict breast cancer gave approximately 40%
of the results as a false negatives. However, by combining the results
from all three assays only one patient appeared as a false negative
(<5%), 71% of patients were scored as strongly positive for breast
cancer (i.e. positive in at least two assays) and 23% of patients were
scored as probable for breast cancer (i.e. positive in at least one
assay). The results also show that a group of patients which have all
been diagnosed with primary breast cancer have different serological
profiles in terms of the immune response to their cancer. Thus, no single
one of the three autoantibody assays would be useful in all primary
breast cancer patients.

Example 6

Cloning of a ras Antigen

Method

[0102] cDNA encoding a mutant oncogenic form of ras (designated K-ras) was
cloned from the cell line KNRK (Rat kidney, Kirsten MSV transformed, see
Aaronson, S. A. and Weaver, C. A. (1971) J. Gen. Virol. 13: 245-252; ATCC
accession number CRL 1569). mRNA was extracted from the cell pellet using
a Dynabead mRNA purification kit according to the manufacturer's
recommended protocol cDNA synthesis, cloning into the EcoRV site of the
PinPoint® vector and transformation of E. coli was carried out as
described in Example 4. Clones expressing ras were then identified by
expression screening using the anti-ras antibody F234-4.2 from
Calbiochem.

Example 7

Cloning of c-myc

Method

[0103] cDNA encoding human c-myc was cloned from the breast cancer cell
line T47-D (European Collection of Animal Cell Cultures accession number
85102201). mRNA was extracted from the cell pellet using a Dynabead mRNA
purification kit according to the manufacturer's recommended protocol.
cDNA synthesis, cloning into the EcoRV site of the PinPoint® vector
and transformation of E. coli was carried out as described in Example 4.
Clones expressing c-myc were then identified by expression screening
using the anti-cmyc antibody 4111.1 from Unilever.

Example 8

Assay for ras and c-myc Autoantibodies

[0104] Biotinylated c-myc and ras antigens were prepared from E. coli
transformed with the appropriate PinPoint® plasmid vector expressing
biotinylated c-myc or biotinylated ras, as described in Example (5), part
(A). The assays for c-myc and ras autoantibodies were then performed
according to the protocol described in Example (5), part (B).

[0105] A group of nine patients previously diagnosed with primary breast
cancer were selected. Pre-operative serum samples were taken from each of
these patients prior to surgery for the removal of the primary breast
cancer. Follow-up serum samples were then taken postoperatively at 2 or 3
monthly intervals and during the same period of time the patients were
assessed clinically for signs of recurrent disease. None of the patients
received any post-operative therapy until recurrence was diagnosed
clinically. The preoperative and post-operative serum samples from each
of the patients were assayed for the presence of autoantibodies to MUC1,
c-erbB2 and p53, using the assay methods described above under Example 5,
and also for the presence of the commonly used serum tumour marker
protein CA15-3. The results of these assays are summarised in Table 3 and
results for three of the nine patients are presented graphically in FIG.
3. Clinical signs of recurrent disease were scored as follows:

[0106] In each of the patients at least one class of autoantibody was
observed to remain above normal level. This suggests continued presence
of the tumour marker (immunogen) and hence continued presence of tumour.
Serum levels of the tumour marker protein CA15-3 were not found to be
predictive of recurrent disease.

Retrospective Analysis of a Well Characterised Series of Healthy Controls
and Patients with Early Breast Cancer

[0107] The above-described methods for detecting autoantibodies to MUC1,
p53, c-erbB2 and c-myc were used to carry out a retrospective study on a
large number of early (stage 1 and 2) breast cancer sera as well as a
large number of control serum samples from individuals with no evidence
of malignancy (control group). The serum samples from patients were all
taken within a 4 week pre-operative period. At the same time, the serum
samples were assayed for the presence of circulating antigen (MUC1 and
c-erbB2) using conventional tumour marker kits (used normally in advanced
disease only). This would allow an assessment of whether the autoantibody
assays are more sensitive than the conventional antigen assays. As used
herein, the terms early or primary breast cancer means that the primary
tumour has a diameter of less than 5 cm. Stage 1 early breast cancer is
defined as lymph node negative; Stage 2 early breast cancer is defined as
lymph node positive.

[0108] In total, pre-operative serum samples from 200 patients diagnosed
with primary breast cancer and 100 normal control samples were assayed
for autoantibodies against MUC1, p53, c-erbB2 and c-myc. The results are
summarised in Tables 4-7 and FIGS. 4-7.

[0109] FIG. 4 depicts the range of autoantibody levels found for each
assay in normal individuals and patients with early breast cancer. It is
apparent that cancer patients have a considerably higher level of
circulating autoantibodies to these markers than do normal individuals.
Using the range for the normal individuals it is possible to set a
`cut-off` above which no normal values should lie. Therefore, samples
with autoantibody levels above this cut-off can be deemed to be positive
for cancer. Cut-off points determined in this manner were used to score
the results of the retrospective study in early breast cancer patients.

[0110] The results presented in Tables 4-7 and FIGS. 5-7 demonstrate the
predictive value of the four autoantibody assays both individually and
when used in combination as a panel of assays. Table 4 indicates the
increased sensitivity of combining the results of a number of assays. By
using one assay on its own, less than 50% of cancers are detected,
however the power of detection increases as more assays are added to the
panel until the combination of all four assays allows 82% of primary
cancers to be detected. FIG. 7 shows the percentage of samples which are
positive in 0 out of 4 assays up to 4 out of 4 assays. This provides good
evidence that the panel assay is more powerful in the detection of cancer
than any one single marker assay since not all patients with cancer have
raised autoantibodies to all markers.

[0111] Tables 5-7 summarise the detection rates in stage 1, stage 2 and in
early breast cancer (i.e. stage 1 and 2) for various combinations of
autoantibody assays. The use of a single autoantibody assay to predict
breast cancer gives approximately 60-70% of the results as false
negatives in the stage 1 group; and 50-60% in stage 2. However, by
combining the results from all four assays, 76% of stage 1 and 89% of
stage 2 cancers were positive in one or more assay. The overall detection
rate for early breast cancer (i.e. both stage 1 and stage 2 cancers)
using this system was 82%. In both stage 1 and stage 2 cancer, assaying
for autoantibodies to MUC1 appeared to add predictive power to any
combination of assays.

[0112] The results for this study were obtained using a 100% confidence
limit, in other words for a result to be deemed positive it had to fall
above the cut-off for readings in the normal range. This normal range was
previously evaluated from a large number of normal individuals and then
confirmed using the control group of 100 normal individuals mentioned
above. Therefore, within the normal control group, none of the samples
were found to be positive, meaning that the sensitivity of the panel of
autoantibody assays was 100% for the detection of early breast cancer
(FIG. 5).

[0113] FIGS. 6 and 7 demonstrate the detection rates which are achievable
if specificity is reduced from a 100% confidence level (no false
positives) to a 95% confidence level, where some degree of false positive
detection is expected. In this case, the cut-off point is defined as the
mean value plus twice the standard deviation of the normal sample range.
Using this cut-off point, approximately 5% of the normal samples were
determined to be positive for cancer (i.e. false positives); whilst
detection of primary cancer increased to approximately 94% (i.e. 6% false
negatives). Again, the greatest percentage of the sample group were
positive in only 1 out of the 4 assays, however, the percentage of
samples that were positive in all 4 assays increased considerably.

[0114] Since the above study was carried out retrospectively, clinical
data was available regarding the initial diagnosis as well as clinical
data regarding the post-operative outcome (i.e. follow-up data). This
allowed analysis of the prognostic value of the data obtained from the
autoantibody assays. Table B shows the correlations between serum levels
of autoantibodies to MUC1, p53, c-erbB2 and c-myc and a number of
clinical factors. For instance, the presence of autoantibodies to any of
the 4 tumour associated proteins (MUC1, p53, c-erbB2 or c-myc) appears to
correlate with the development of a recurrence. In other words, those
patients who had autoantibodies were more likely to go on to develop a
recurrence of their disease. In the case of autoantibodies to MUC1, c-myc
and c-erbB2, this was most likely to be distant metastases, only
autoantibodies to p53 were not associated with the later development of
distant metastases with any statistical significance. In fact, the
presence of autoantibodies to p53 was the weakest indicator of a later
recurrence of disease; furthermore, p53 autoantibodies correlated with
disease free interval.

[0115] Table 9 presents an analysis of whether the degree of autoantibody
positivity may be of value in the prediction of which stage 1 tumour will
go on to develop a recurrence. At the present time, there is little to
indicate at the time of diagnosis whether a patient with a stage 1 tumour
(i.e. no evidence of spread of tumour to the lymphatic system) will go on
to develop recurrent disease. As can be seen in Table 9, of those
patients with stage 1 tumours from the sample group that went on to
develop recurrent disease, 71% were positive in two or more autoantibody
assays. Of the patients with stage 1 tumours that have not yet recurred,
only 30% were positive in two or more autoantibody assays.

Detection of Autoantibodies in Sequential Serum Samples--Application to
the Monitoring of Disease Progression

[0116] This study was carried out in order to assess whether autoantibody
assays could be useful in the earlier detection of recurrent disease.

[0117] Levels of autoantibodies to MUC1, p53 and c-erbB2 in the serum of
patients previously diagnosed with breast cancer were measured
sequentially during follow-up until the patient manifested recurrent
disease. The results are summarised in FIGS. 8-10. All three patients
went on to develop recurrent disease. In all three patients, autoantibody
levels were indicative of the presence of cancer. However, there is no
evidence from this group that autoantibody levels decrease after removal
of the primary tumour. FIG. 10 shows the levels of autoantibodies
post-operatively of a patient with non-recurrent disease and a patient
with recurrent disease. Autoantibody levels in the patient with
non-recurrent disease remained below the cut-off point during the period
of sample collection (48 months). In the second patient, whose disease
recurred at 36 months, autoantibody levels are seen to be steadily rising
towards the cut-off point, with c-erbB2 autoantibodies rising above
cut-off. Furthermore, as can be seen in FIG. 9, when further sequential
samples are added to the analysis, 3 out of the 4 assays become positive
for cancer and these levels then decrease again once treatment of the
recurrence is underway. This data supports the utility of autoantibody
assays in the earlier detection of recurrent disease.

Example 12

Analysis of a Series of Patients with Bladder Cancer and Benign Urological
Disorders

[0118] Serum samples were collected from a group of 80 patients with
bladder cancer/benign urological disorders and analysed for the presence
of autoantibodies to MUC1, p53, c-erbB2 and c-myc using the assay methods
described above.

[0119] The data summarised in Table 10 shows that single assay
sensitivities for bladder cancer detection range from 15-50% (as opposed
to 35-47% for breast cancer). The detection sensitivity using all 4
assays was 80%, similar to that found for early breast cancer.

[0120] FIG. 11 shows the break down of detection rates between
urologically benign disorders (`benign`) and the three stages of bladder
cancer. Upon further investigation of the relevant clinical data it
became apparent that 6 of the patients in the `benign` group had evidence
of other malignancies. These other malignancies were lung cancer, skin
cancer and adenocarcinoma. Evidence of other malignancies were: pleural
effusion, ovarian cysts and colon polyps. Serum samples from all 6 of
these patients had been scored as positive for cancer using the panel of
autoantibody assays, illustrating the general application of the panel
assay to the detection of cancers. Furthermore, it is known that some
patients with stage PT1/2 and PT3/4 disease had previously received
systemic therapy.

[0121] An autoantibody assay as previously described was carried out on
serum samples from patients with colorectal cancer using the tumour
antigens c-myc, p53, c-erbB2 and K-ras individually and as a panel. The
results are shown in FIGS. 12 and 13. As has been demonstrated previously
increased sensitivity is shown when a panel of antigens is used.

Example 14

Use of BRCA1 in Panel Assay for Detection of Breast Cancer

[0122] A BRCA1 antigen suitable for use in the detection of anti-BRCA1
autoantibodies was cloned from the breast cancer cell line MCF7 using an
RT-PCR strategy. Briefly, mRNA isolated from MCF7 cells was reverse
transcribed to give first-strand cDNA. These cDNA was used as a template
for PCR using a primer pair designed to amplify a product covering the
first 1500 base pairs of the BRCA1 cDNA but including a known mis-match
mutation that leads to an early stop codon and therefore the production
of truncated protein. Different sites for restriction enzyme digestion
were also incorporated into the forward and reverse PCR primers to
facilitate the cloning of the PCR product. The PCR primers were as
follows:

[0123] The PCR product obtained using these primers was then cloned into
the PinPoint® vector and used to transform E. coli Top 10 F cells, as
described hereinbefore. Clones expressing the fusion protein of truncated
BRCA1 antigen fused in-frame to the N-terminal biotinylation domain were
then identified by expression screening, according to the procedure
described in Example 4, using the antibody MAB4132 from Chemicon.

[0124] Biotinylated truncated BRCA1 antigen is then prepared from E. coli
transformed with the appropriate PinPoint® plasmid vector expressing
the fusion protein, as described in Example (5), part (A). The assay for
BRCA1 autoantibodies is then performed according to the protocol
described in Example (5), part (B).

[0125] FIG. 14 shows the results of a study in which the above-described
assays for autoantibodies to cmyc, p53, c-erbB2, MUC1 and BRCA1 were
performed individually, as a panel and as a panel without BRCA1 to detect
autoantibodies in samples of serum taken from normal individuals,
patients diagnosed with primary breast cancer and BRCA1 mutation
carriers. As demonstrated previously, increased sensitivity is shown when
a panel of markers is used.

[0126] cDNA encoding human PSA was cloned from the cell line T47-D using a
protocol similar to that described above for the cloning of c-erbB2.
Briefly, the T47-D cells were first stimulated with Apigenin at 10-5M as
described by Rosenberg et al. (1998) Biochem Biophys Res Commun. 248:
935-939. mRNA was then extracted and cDNA synthesis, ligation into
PinPoint® and transformation of E. coli. performed as described in
Example 4. Clones expressing PSA were identified using an anti-PSA
antibody. Biotinylated PSA antigen was prepared from E. coli transformed
with the PinPoint® vector expressing biotinylated PSA according to the
protocol described in Example (5), part (A). The assay for PSA
autoantibodies was then performed according to the protocol described in
Example (5), part (B).

[0127] An autoantibody assay using the methods described above was carried
out on patients with prostate cancer using c-myc, p53, c-erbB2, PSA and
MUC 1 individually and as a panel. The results are shown in FIG. 15 and
confirm the increased sensitivity of such a panel for detection of
prostate cancer.

Example 16

Other Tumour Marker Antigens

[0128] CA125 can be affinity purified from the ovarian cancer cell line
OVRCAR-3 (available from the ATCC) using Mab VK-8, as described by Lloyd,
K. O. et al. (1997) Int. J. Cancer. 71: 842-850.

[0129] APC protein is expressed by the colorectal cancer cell line SW480
(available from the ATCC) as described by Munemitsu, S. et al. (1995)
PNAS 92: 3046-3050.